Docosapentaenoic acid is one of rare unsaturated fatty acids, which is C22:5 n-3, contained in fish oil by a slight amount. Mass production of highly pure docosapentaenoic acids is strongly expected for the promotion of physiological/medical/nutritional research (Non Patent Literature 1).
With regard to this point, the following facts have been reported (Non Patent Literature 2).    (1) Docosapentaenoic acid is generated from icosapentaenoic acid in many tissues, and the opposite occurs at the same time.    (2) Docosapentaenoic acid is effective in lowering triglyceride levels.    (3) Docosapentaenoic acid is metabolized in blood platelets to give hydroxydocosapentaenoic acid. Starting with this fact, facts that docosapentaenoic acid is converted in vivo into resolvin D4, which is one of lipid mediators, and this activates the immune system to exhibit anti-inflammatory action, have been made clear one after another.    (4) Docosapentaenoic acid is effective in maintaining and promoting health.
Non Patent Literature 3 reports the following.    (5) The rabbit platelet agglutination inhibitory action of docosapentaenoic acid is stronger than icosapentaenoic acid or docosahexaenoic acid, and a clot formation suppressing effect can be expected.    (6) Docosapentaenoic acid also has an endothelial cell migration capability that is 10-times greater than icosapentaenoic acid. This is an important effect in wound healing.
Non Patent Literature 4 reports the following.    (7) The action of lowering fatty acid synthase and malate synthase activity of docosapentaenoic acid is stronger than icosapentaenoic acid.    (8) Docosapentaenoic acid possibly regulates a phenomenon of sustained improvement in signaling between two nerve cells by aging-related spatial learning and costimulation.
Non Patent Literature 5 reports the following.    (9) Docosapentaenoic acid exerts an angiogenesis suppressing effect.
As discussed above, docosapentaenoic acid would play an important role in fields of alternative medicines and health food in the future, and the demand thereof would accordingly increase. Thus, development of a highly efficient production method by the present invention is an urgent problem to be solved. In recent years, biological functions of polyunsaturated fatty acids, especially icosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid from fish oil, are gaining increased attention. The demand thereof has been increasing, including high-purity icosapentaenoic acid as a pharmaceutical. In addition, the demand for polyunsaturated fatty acids as supplements such as docosahexaenoic acid having action of improving cranial nerve functions is also expanding. On the other hand, polyunsaturated fatty acid resources are in a declining trend on a global scale, and thus securement of those acids has been explored worldwide as an important problem to be solved. Currently, they mostly depend on fishery resources which are mainly fishes. However, a research related to a method of production using algae and plants is actively pursed. For instance, Monsanto Company established a method of producing stearidonic acid, which is a precursor of icosapentaenoic acid, using genetically modified soybeans, and this is already approved by the FDA. In addition, methods of using a chemical reaction to extend polyunsaturated fatty acids or a microbially produced desaturase have also been reported. However, it is generally difficult to simultaneously conduct reactions of carbon chain extension on polyunsaturated fatty acids in a large scale, and thus said method is not at a stage where it can be practiced in a practical production level beyond a laboratory scale. A synthesis method of polyunsaturated fatty acids by a chemical reaction of carbon chain extension that does not use an enzyme has been reported. For example, in Non Patent Literature 6, one methylene proton in a p-toluenesulfonylmethyl isocyanate molecule is abstracted with a base and the produced carbanion is reacted with saturated fatty acid methyl ester bromide to synthesize isocyanate having a carbon long chain, and a strong base such as sodium hydride is used to similarly replace the other proton of the methylene chain with an unsaturated chain. Lastly, lithium/ammonia/ethanol and methanol/hydrochloric acid are reacted to perform a reaction of removing toluenesulfonyl and a reaction of removing isonitrile to synthesize a polyunsaturated fatty acid methyl ester of interest. However, these methods are disadvantageous in that the total yield is low, and an expensive reagent or a reagent that has strong reactivity and is difficult to handle, must be used.
Meanwhile, Baba et al. have successfully synthesized tetracosahexaenoic acid methyl ester with 2 more carbon atoms than docosahexaenoic acid methyl ester through: using docosahexaenoic acid ethyl ester as a starting material to produce alcohol by lithium aluminum hydride reduction and converting the alcohol into p-toluenesulfonic ester; then converting the p-toluenesulfonic ester into iodide by a substitution reaction; in the presence of a base, further reacting diethylmalonate therewith to produce malonate ester; and subjecting the ester to alkaline hydrolysis, decarboxylation, and methyl esterification (Non Patent Literature 7 and Non Patent Literature 8).
This reaction process was also applied to a carbon chain extending reaction of linoleic acid or arachidonic acid. However, since multistage reaction steps are included, this is not necessarily considered as a practical method for large-scale production.
The method published by Ito et al. in 2011 synthesizes tetracosahexaenoic acid with 2 more carbons in 4 steps from docosapentaenoic acid ethyl ester (Non Patent Literature 9). However, this method uses an explosive reagent that is very unstable to air called DIBAL-H, and a reaction is performed at a low temperature of −78° C. Thus, it is expected that scaling up of the synthesis process would be difficult.
According to references in the 1970s (Non Patent Literatures 10 and 11), a method of converting fatty alcohol into methanesulfonic ester, and introducing diethyl malonate into this has been reported. By employing this method, synthesis of iodide as an intermediate becomes unnecessary, and it is possible to omit one step from the full process. However, in the reaction example carried out in these references in the 1970s, a heating reaction is performed by using high boiling point solvents such as xylene and butanol in a closed system where pressure is applied. Such a reaction condition is not suitable for carbon chain extension of EPA, DPA and DHA, which are unstable to heat. In the present patent, by using dimethylformamide or dimethyl sulfoxide, which is a polar aprotic solvent that activates carbon anion, instead of xylene and butanol, it is possible to synthesize malonate anion having high reactivity more efficiently, and by using this, it is possible to easily synthesize malonic ester derivatives also from methanesulfonic esters of alcohols obtained from EPA, DPA and DHA. By employing this step, it is possible to synthesize DPA EE from EPA EE in more shortened 4 steps.